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. 2013 Feb 7;33(2):e00023. doi: 10.1042/BSR20120104

Figure 1. TPP-1 activity in control fibroblasts, LINCL cells and LINCL loaded with recombinant TPP-1.

Figure 1

TPP-1 activity was measured in CTL and LINCL cells pre-incubated in the presence (LINCL+TPP-1) or in the absence (LINCL) of 10 nM recombinant TPP-1 for 48 h using the synthetic substrate Ala–Ala–Phe–AMC (AAF–AMC). Briefly, at the end of the incubation with the recombinant enzyme, cells were maintained for 72 h in culture with fresh medium without TPP-1. Cells were then homogenized in ice-cold isotonic sucrose and membranes were solubilized with Triton X-100. TPP-1 activity was assayed in 50 mM acetate buffer (pH 5.0) containing 5 mM 4-methylumbelliferyl derivative as substrate. The fluorescence of 4-methylumbelliferone released was measured in a spectrofluorimeter (λexc, 350 nm; λem, 460 nm). Results were calculated as arbitrary fluorescence units normalized for protein content (AFU/μg of proteins) and expressed in percentages of CTL cells as means±S.D. (n=3). **, ***: significantly different from CTL cells with P<0.01 and P<0.001 respectively. ###: significantly different from LINCL cells loaded with TPP-1 with P<0.001 as determined by an ANOVA-1 followed by a Dunnett's test.